In this project, the applicant will investigate the regulation of Na+/Ca2+ exchange activity in transfected Chinese hamster ovary cells that permanently express either the wild-type cardiac Na+/Ca2+ exchanger or mutant exchangers created by site-directed mutagenesis. The cental hypothesis is that exchange activity in intact cells is regulated at three different levels of complexity: (1) throughkinetic alterations induced by changes in cytosolic (Ca2+), (2) through interactions with the cytoskeleton, and (3) through interactions with Ca2+ sequestering organelles. The applicant will characterize the regulation of exchange activity by cytosolic (Na+), (Ca2+), and pH by measuring exchange-mediated Ba2+ influx in cells treated with the ionophores gramicidin and ionomycin. The applicant will also examine the influence of exchange activity of cytoskeletal changes induced by the phosphatase inhibitor calyculin A, and by osmotic shrinkage, with special emphasis on the role of intermediate filaments in the response. Finally, the applicant will use selective inhibitors and Ca2+ leading/depletion protocols to examine the role of Ca2+ uptake and release by the endoplasmic reticulum and otehr Ca2+ sequestering organelles in activating or deactivating exchange activity.